Preparation of Nanometric Arrays of Biomolecules on Oligo-or Poly(Ethylene Glycol) Films on Silicon Surfaces

ABSTRACT

The present invention is generally directed to nanometric biomolecular arrays and to a novel approaches for the preparation of such nanoarrays, based on binding of biomolecules, such as avidin, to templates generated by lithographically-an-odizing biocompatible ultrathin films on silicon substrates using AFM anodization lithography. The present invention is also directed to methods of using such arrays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims priority to U.S. Provisional Application Ser.No. 60/566,120, filed Apr. 28, 2004.

This invention was made, in part, with support from the National ScienceFoundation, Grant Number CTS-0210840; and the Robert A. WelchFoundation, Grant Number 1-5-57897.

FIELD OF THE INVENTION

This invention relates generally to microarrays, and specifically tonanometric biomolecular arrays and their fabrication.

BACKGROUND OF THE INVENTION

Microarray technology has been widely used for genomics and proteomicsresearch as well as for drug screening. Currently, the spot size in mostmicroarrays is larger than one micron. The use of nanometricbiomolecular arrays, with smaller spot sizes, will enablehigh-throughput screening of biomolecules—eventually at the singlemolecule level. Also, nanometric arrays permitting precise control overthe position and orientation of individual molecules will become apowerful tool for studying multi-valent and/or multi-component molecularinteractions in biological systems. Toward these ends, protein arrayswith feature sizes smaller than 100 nm have been fabricated, mostlyusing dip-pen nanolithography and nanografting. See Lee et al., Science2002, vol. 295, p. 1702; Wilson et al., Proc. Natl. Acadi Sci. USA 2001,vol. 98, p. 13660; Liu et al., Proc. Natl. Acad. Sci. USA 2002, vol. 99,p. 5165; Pavlovic et al., Nano Lett. 2003, vol. 3, p. 779; and Krämer etal., Chem. Rev. 2003, vol. 103, p. 4367.

Biological microelectromechanical systems (bioMEMS) are of tremendousinterest for their potential applications in microscale, high throughputbiosensing and medical devices (Shawgo et al., J. Curr. Opin. SolidState Mater. Sci. 2002, v. 6, p. 329). Using silicon as a substrate forthe preparation of such devices is particularly attractive, since theextensive micro-fabrication techniques developed by the microelectronicindustries can be used to fabricate and integrate variousmicro-components into the devices. For reducing biofouling, considerableresearch has been directed to the modification of substrate surfaceswith stable and ultrathin films of poly(ethylene glycol) (PEG) oroligo(ethylene glycol) (OEG) (Prime et al., Science 1991, vol. 252, p.1164). Since many of the ultimate applications for bio-devices requiremoderate-term (e.g., a few hours to several days) exposure to biologicalmedia (e.g., buffer of pH 7.4 at 37° C.), stability of thebio-compatible coatings on the devices under these conditions is highlydesirable. All of the OEG/PEG-terminated films on silicon substratesreported by others are bound onto the silicon surfaces via Si—O bondsthat are prone to hydrolysis (Calistri-Yeh et al., Langmuir 1996, v. 12,p. 2747), thereby limiting their stability under physiologicalconditions (Sharma et al., Langmuir 2004, v. 20, p. 348).

As described in commonly assigned, co-pending U.S. patent applicationSer. No. 10/742,047, olig(ethylene glycol) (OEG) terminated alkenes weregrafted onto hydrogen-terminated silicon surfaces throughhydrosilylation (as developed by Linford and Chidsey, see Linford etal., J. Am. Chem. Soc. 1993, v. 115, p. 12631; Buriak, Chem. Rev. 2002,v. 102, p. 1271) forming robust Si—C bonds with the silicon surfaces. Itwas shown that the alkyl monolayers grown by this method were stable inboiling organic solvents, water, and acids, as well as slightly basicsolutions (Linford et al., J. Am. Chem. Soc. 1995, v. 117, p. 3145). Amethod describing the modification of hydrogen-terminated siliconsurfaces, including a silicon atomic force microscopy (AFM) cantilevertip, with OEG-terminated alkenes via either thermally- or photo-inducedhydrosilylation is also found in commonly assigned, co-pending U.S.patent application Ser. No. 10/742,047. See also Yam et al., J. Am.Chem. Soc. 2003, v. 125, p. 7498; Yam et al., Chem. Commun., 2004, p.2510). The efficiency with which such OEG-terminated films resistprotein adsorption depends on many factors including the number ofethylene glycol (EG) units and the packing density of the films that isdetermined by the underlying substrate surface and the depositionmethods. For example, OEG-terminated thiolate self-assembled monolayers(SAMs) on gold (111) surfaces are protein resistant, but those on silver(111) surfaces are not (Herrwerth et al., J. Am. Chem. Soc. 2003, vol.125, p. 9359). The latter was attributed to the high packing density andstructural ordering of the SAMs. Research has demonstrated that filmsgrown on Si (111) surfaces had a density similar to that of thecorresponding thiolate SAMs on gold (111) surfaces, and similarlyreduced the adsorption of fibrinogen to 1% monolayer or less (Cai etal., to be submitted).

As a result of the foregoing, a nanometric biomolecular array comprisinga stable, pattemable monolayer surface, and an efficient method ofmaking such an array, would be very beneficial.

BRIEF DESCRIPTION OF THE INVENTION

The present invention is generally directed to a nanometric biomoleculararray, and to methods of making such arrays, wherein such methodstypically involve AFM anodization lithography. The present invention isalso directed to methods of using such arrays.

In some embodiments, such above-mentioned methods generally comprise thesteps of: (a) contacting OEG-terminated alkenes with ahydrogen-terminated Si surface to form a contacted surface; (b)photolyzing the contacted surface to effect Si—C bonding between theOEG-terminated alkenes and the Si surface and form an OEG-coated Sisurface comprising a monolayer of OEG bound to the Si surface throughSi—C bonds; (c) anodizing regions on the top of the OEG monolayer of theOEG-coated Si surface via AFM anodization lithography to yield ananolithographically-patterned OEG-coated Si surface comprising regionswith enhanced associability toward biomolecules; and (d) depositing atleast one type of biomolecule in the regions of enhanced associabilityto form a nanometric biomolecular array.

Generally, the nanometric biomolecular arrays made by theabove-described methods comprise: (a) a Si substrate; (b) a monolayer ofOEG bonded to the Si substrate via Si—C bonds, wherein regions at thetop of the monolayer have been lithographically-patterned; and (c)biomolecules associated with the lithographically-patterned regions ofthe OEG monolayer.

In some embodiments, the present invention provides a novel approach forpreparation of uanometric protein arrays, based on binding ofbiomolecules to nano-templates generated by AFM anodization lithographyon robust, ultrathin monolayers terminated with oligo(ethylene glycol)(OEG) derivatives with the general formula of —(CH₂CH₂O)_(n)—R (n>1,R═CH₃, H, etc.) on conducting silicon surfaces. A specific example isthe preparation of nanometric avidin arrays. Applicants have shown thatbiotinated-BSA, but not the native BSA, binds to the avidin arrays, andthe resulting arrays of biotinated-BSA can bind avidin to form proteindots with a feature sizes of ˜30 nm, scalable down to the size of asingle protein molecule.

Such nanometric arrays have at least the following unique advantages:(a) they will vastly improve the detection sensitivity (down to a singlemolecule), allowing for detection of biomolecular variations correlatedwith diseases, which are typically expressed at very low level; (b) theywill tremendously increase the probe density on a chip (e.g.,incorporating the whole human genome in the same chip); (c) they permita label-free detection of the binding of target molecules on thenanoarrays; (d) they greatly shorten the time for binding of targetmolecules to the nanoarrays and improve the efficiency of the binding;(e) they may substantially improve the specificity of the detection; (f)the single molecule arrays will greatly facilitate single moleculesequencing of DNA using polymerase and nucleotides that arefluorescently labeled, and single molecule arrays of the template andthe polymerase will reduce the background fluorescence and greatlyimprove the quality of the data; and such (g) nanometric arrays willbecome a powerfuil research tool for studying the cooperativeinteraction among multiple biomolecules. It should be noted thatadvantages (a)-(e) can be gained only for nanoarrays where the spot sizeis less than about 25 nm and comprising only one or a handfuil of probemolecules.

The foregoing has outlined rather broadly the features of the presentinvention in order that the detailed description of the invention thatfollows may be better understood. Additional features and advantages ofthe invention will be described hereinafter which form the subject ofthe claims of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, and theadvantages thereof, reference is now made to the following descriptionstaken in conjunction with the accompanying drawings, in which:

FIG. 1 depicts, schematically, a method of making nanometricbiomolecular arrays, in accordance with embodiments of the presentinvention;

FIG. 2 depicts height (2 a, 0.5×0.5 μm², 10 nm contrast) and friction (2b, 0.1 V contrast) AFM images of an EG₇ film on Si (111) after AFManonization lithography, and a 3-dimensional image (2 c, 5×5 μm²) of apatterned area upon treatment with succinic anhydride, DMAP andpyridine;

FIG. 3 depicts AFM height (3 a, 3 c, and 3 e) and friction (3 b and 3 d,corresponding to 3 a and 3 c) images (4×4 μm²) of an area similar tothat shown in FIG. 2 c upon sequential treatment with EDAC/avidin (3a-b), biotinated-BSA (3 c-d), and avidin (3 e), wherein the lines in 3a-b are used as guides;

FIG. 4 demonstrates, schematically, a nanofabrication process, inaccordance with embodiments of the present invention;

FIG. 5 illustrates a setup for AFM anodization lithography, inaccordance with embodiments of the present invention; and

FIG. 6 illustrates AFM anodization lithography of an OEG monolayer on aSi surface, wherein the patterned (anodized) regions comprise functionalmoieties such as carboxylic acid, aldehyde, and/or alcohol, etc.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is generally directed to nanometric biomoleculararrays and to a novel approaches for preparation of such nanoarrays,based on binding of biomolecules, such as avidin, to templates generatedby AFM anodization lithography (conductive AFM) on biocompatibleultrathin films on silicon substrates. In some embodiments, such filmsare generally robust, ultrathin monolayers terminated witholigo(ethylene glycol) (OEG) with the general formula of—(CH₂CH₂O)_(n)—R (n>1, R═CH₃, H, etc.) on conducting silicon surfaces,wherein such films have been nanolithographically-patterned usingconductive AFM lithography (Maoz et al., Adv. Mater. 2000, vol. 12, p.725). The lithography process is followed by chemical and biochemicalderivatization of the resulting nanopatterns. The unique features ofthis approach include: (a) the OEG-monolayers resist non-specificadsorption and denaturing of proteins on the templates; (b) conductiveAFM can be used to selectively oxidize the top portion of theOEG-monolayer to generate carboxylic acids, aldehydes, alcohols andother functional groups that can be used to attach biomolecules; (c) themonolayers are attached to silicon substrates via Si—C bonds with a highdensity, rendering the system highly robust; and (d) the lithographyprocess is very rapid. In a specific example, the resulting avidinarrays have a feature size of ˜26 nm, and they can serve as templatesfor the preparation of nanoarrays of a wide variety of proteins that aresite-specifically labeled with biotin (Lue et al., J. Am. Chem. Soc.2004, v. 126, p. 1055).

Referring to FIG. 1, in some embodiments, such above-mentioned methodsgenerally comprise the steps of (Step 101) contacting OEG-terminatedalkenes with a hydrogen-terminated Si surface to form a contactedsurface; (Step 102) photolyzing the contacted surface to effect Si—Cbonding between the OEG-terminated alkenes and the Si surface and form aOEG-coated Si surface comprising a monolayer of OEG bound to the Sisurface through Si-C bonds; (Step 103) lithographically anodizingregions on the top of the OEG monolayer of the OEG-coated Si surface viaAFM anodization lithography to yield a nanolithographically-pattemedOEG-coated Si surface comprising regions with enhanced associabilitytoward biomolecules; and (Step 104) depositing at least one type ofbiomolecule in the regions of enhanced associability to form ananometric biomolecular array.

In some embodiments, the OEG-terminated alkenes comprise EG sequencesselected from the group consisting of EG₁-EG₂₀, and combinationsthereof, wherein “n” in EG_(n) describes the number of —(CH₂CH₂O)—repeat units. In some or other embodiments, the OEG-terminated alkenescomprise PEG-terminated alkenes, wherein PEG-terminated alkenes compriseEG_(n) sequences of n>20.

Typically, the Si surface is atomically flat. In some embodiments the Sisurface is selected from the group consisting of (100), (111), andcombinations thereof In some embodiments, upon coating the Si surfacewith an OEG monolayer, the OEG-coated Si surface is washed, andoptionally dried, prior to lithographically anodizing regions on top ofit.

In some embodiments, the nanolithographically-patterned (anodized)regions of the OEG-coated Si surface comprise nanowells (i.e., “spots”).In some embodiments, the nanolithographically-patterned regions of theOEG-coated Si surface comprise carboxylic acid, aldehyde, alcohol,and/or other moieties, wherein these moieties provide, at least in part,the enhanced associability toward biomolecules.

In some embodiments, the at least one type of biomolecule is selectedfrom the group consisting of proteins, oligonucleotides, andcombinations thereof Avidin is an exemplary such biomolecule. In someembodiments, at least some of the at least one type of biomolecule bindswith the regions of enhanced associability via amide bonds.

Generally, the nanometric biomolecular arrays made by theabove-described methods comprise: (a) a Si substrate; (b) a monolayer ofOEG bonded to the Si substrate via Si—C bonds, wherein regions at thetop of the monolayer have been lithographically-patterned; and (c)biomolecules associated with the lithographically-patterned regions ofthe OEG monolayer.

Typically, the above-mentioned Si surface is atomically flat. In someembodiments, the Si surface is selected from the group consisting of(100), (111), and combinations thereof In some embodiments, the OEGbound to the Si surface comprises EG sequences selected from the groupconsisting of EG₁-EG₂₀, and combinations thereof As mentioned above,such OEG is bound to the surface through Si—C bonds.

In some embodiments, the biomolecules (as part of the array) areselected from the group consisting of proteins, oligonucleotides, andcombinations thereof Avidin is an exemplary such molecule. Typically,the biomolecules are associated by a bonding means selected from thegroup consisting of covalent bonding, ionic bonding, electrostaticforces, and combinations thereof In some particular embodiments, thebiomolecules are associated with the lithographically-patterned regionsof the OEG monolayer via amide bonds.

In some embodiments, the nanometric biomolecular array is operable forbinding biomolecular analyte, i.e., it can be used to assay biomolecularanalyte, wherein biomolecular analyte can comprise one or more of avariety of different biomolecules. In such embodiments, biomolecularanalyte is deposited on the array, and the array is analyzed todetermine the regions in which the biomolecular analyte exhibits abinding affinity. In some such embodiments, the biomolecules andbiomolecular analyte are removed by treatment with proteinase K, whereinthe proteinase K serves to catalyze hydrolytic fragmentation of proteinsbound to the patterned OEG monolayer surface to regenerate the pattern.Biomolecular analyte suitable for such analysis (including sequencing)include, but are not limited to, oligonucleotides, proteins, andcombinations thereof In some or other embodiments, such an array isuseful for screening drug candidates.

The monolayers described herein can be readily prepared fromα-hepta-(ethylene glycol) methyl ω-undecenyl ether, comprising seven EG(EG₇) repeat units (the term “EG₇” being used herein to describe boththe repeat units and the alkene precursor comprising the seven EG repeatunits), and conductive silicon (111) substrates with an atomically-flat,H-terminated surface (Yam et al., Chem. Commun., 2004, p. 2510). Inaccordance with some embodiments, AFM anodization lithography on thesemonolayers was performed under ambient conditions with a relativehumidity of ˜20-80%, using a Nanoscope IIIa AFM (Digital Instrument)equipped with a pulse generator. During AFM anodization on eachlocation, a short pulse of +(4 to 17) V, with a duration typically inthe range of about 10 nanoseconds (ns) to about 10 microseconds, wasapplied to the sample while the tip was grounded During AFM anodization,the AFM scanner can rapidly position the AFM tip on the sample withnanometer precision. This process for generating a high-densitynanoarray proved to be much faster than dip-pen or nanograftinglithography techniques that normally take seconds to generate eachnanospot. The pulse generator was then disconnected, and height andfriction AFM images were simultaneously acquired in contact mode at a90° scan angle with the same tip. In one specific example, in which thenanolithography was performed with 100 ns pulses of +10 V, it was foundthat holes with an apparent depth of 0.4 nm (corresponding to the lengthof one ethylene glycol unit) were generated, as revealed by the heightimages of the patterned areas, e.g., FIG. 2 a However, the correspondingfriction image (FIG. 2 b) shows the presence of spots of ˜10 nm indiameter where the friction is higher than the surroundings, indicatingthe presence of polar head groups on the spots. Referring to FIG. 2, thespacing between the spots was ˜50 nm, as controlled by the scanner.

While not intending to be bound by theory, it has been suggested thatAFM anodization of alkyl siloxane monolayers on silicon under certainconditions could oxidize the head groups of the monolayers intocarboxylic (COOH) groups (Maoz et al., J. Adv. Mater. 2000, v. 12, p.725). However, a recent study of a similar system using secondary ionmass spectroscopy showed no signs of COOH groups on the oxidizedsurfaces (Pignataro et al., Mater. Sci. Eng. C. 2003, v. 23, p. 7).

It should be noted that the monolayers in the above-described study hadalkyl head groups, while the monolayers of the present inventioncomprise an OEG head group. The AFM anodization of the OEG-coatedsurfaces could be substantially different from the above-descnrbed alkylsurfaces. Preliminary studies by Applicants indicate that, upon AFManodization of OEG-coated surfaces, a variety of species includingcarboxylic acids, aldehydes, and alcohols, are generated on the filmsurface. Additionally, in some embodiments, treatment of an AFM-anodizedmonolayer with avidin is done in the presence of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC, which serves tomediate the formation of amide bonds between the surface COOH groups andthe protein molecules). Corresponding AFM images obtained during thefirst few scans showed that the protein molecules predominately adsorbedon the patterned spots. The protein molecules were readily removed bythe scanning tip afterwards indicating that the protein molecules werenot covalently bound to the surface. Again, while not intending to bebound by theory, it was concluded that rather than COOH groups, thesurfaces of the oxidized spots mainly comprised hydroxyl groups thatcould be chemically converted into COOH groups, e.g., by treatment withsuccinic anhydride, dimethyl-aminopyridine (DMAP) and pyridine.Patterned spots were “etched” upon this treatment forming nanoholes asshown by the three-dimensional AFM height image (FIG. 2 c). As measuredby the line profile of about 100 patterned spots in FIG. 2 c, thediameter of the holes was 91±6 nm, and the depth was 1.31±0.12 nm, aboutone third of the thickness of the film. Using the method describedherein, COOH groups were generated in the nanoholes, which may be usedto attach proteins. Upon incubation of the samples with EDAC followed byavidin in PBS solution, the nanoholes were nearly filled (FIG. 3 a) andbarely recognized even by comparison with the corresponding frictionimage (FIG. 3 b). The depth of the holes decreased to 0.43±0.06 um,while the width of the holes remained nearly the same (87±9 nm).

The sample was then treated with BSA in PBS buffer. The depth of theholes remained the same, indicating that BSA did not bind to themolecules in the holes. To verify that the molecules in the holes wereindeed avidin, the sample was treated with a solution of biotinated-BSAin PBS buffer. AFM height and friction images (FIG. 3 c and 3 d) revealthat the patterned spots protrude slightly from the film surface. Theheight and half-height width of the spots were 0.14±0.14 nm and 24±3.5nm, respectively. The fact that the molecules in the holes boundbiotinated-BSA but not native BSA is a strong indication that thesemolecules were avidin.

The patterned biotinated-BSA, with an average of nine biotin groups oneach BSA molecule, should have free biotin groups available for bindingadditional avidin onto the pattern. Indeed, upon incubation of thesample in a solution of avidin in PBS, nano-dots arrays were formed, asshown by the AFM height image (FIG. 3 e). The heights of the dots were1.27±0.37 nm and the half-height widths of the dots were 26±3.4 nm.While the top avidin molecules could be removed by repeated scanning,the protein molecules in the holes were strongly bound, and could not beremoved by the scanning tip, neither by immersion in PBS for 6 hours norin detergent (SDS) solutions for 14 hours. AFM images of the proteinarrays remained nearly the same after four weeks under ambientconditions. Upon treatment with Proteinase K (to catalyze the hydrolyticfragmentation of the proteins), nanoholes very similar to those in FIG.2 c were regenerated. The nanofabrication process, in accordance withsome embodiments of the present invention, is demonstrated in FIG. 4.

Nanometric biomolecular array fabrication, as described herein and inaccordance with embodiments of the present invention, will vastlyimprove the detection sensitivity (down to a single molecule), greatlyfacilitate single molecule sequencing of DNA, and serve as a powerfulresearch tool for studying the cooperative interaction among multiplebiomolecules.

The following examples are provided to demonstrate particularembodiments of the present invention. It should be appreciated by thoseof skill in the art that the methods disclosed in the examples whichfollow merely represent exemplary embodiments of the present invention.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments described and still obtain a like or similar result withoutdeparting from the spirit and scope of the present invention.

EXAMPLE 1

This example serves to illustrate materials used in the fabrication ofnanometric protein arrays, on protein-resistant monolayers on siliconsurfaces, in accordance with embodiments of the present invention.

Pyridine, succinic anhydride, 4-dimethylaminopyridine (DMAP),1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC), avidin, bovineserum albumin (BSA), biotinamidocaproyl labeled BSA (biotin-BSA),proteinase K, and phosphate buffered saline (PBS buffer, 0.01 Mphosphate, 0.14 M NaCl, pH 7.4) were purchased and were used withoutpurification.

EXAMPLE 2

This example serves to illustrate the synthesis of hepta(ethyleneglycol) methyl ω-undecenyl ether (comprising EG₇), as used in someembodiments of the present invention.

Monomethyl hepta(ethylene glycol) (1.637 g, 4.81 mmol) was slowly addedto NaH (0.81 g, 33.75 mmol) in dry THF (8 ml) while stirring under N₂.To this mixture was added Bu₄NI (0.81 g, 0.48 mmol) and11-bromo-1-undecene (4.2 ml, 16.78 mmol), and the mixture was refluxedfor 20 hours under N₂. Iodomethane (3.42 g, 24.1 mmol) was added, andthe mixture was refluxed for 1 hour. The reaction mixture was thenrefluxed with methanol for another hour. After cooling to roomtemperature, the mixture was concentrated under reduced pressure.Dichloromethane was added, and the mixture was subsequently poured intowater. The organic layer was separated, and the aqueous layer wasextracted twice with dichloromethane. The combined organic layers werewashed twice with water, dried with magnesium sulfate, filtered andconcentrated under reduced pressure. The crude product was purified byflash chromatography (ethyl acetate/hexane/methanol 50:48:2) to affordEG₇ (1.8 g, 76%). ¹H NMR (CDCl₃): δ=5.80-5.82 (m, 1H); 4.90-5.01 (m,2H); 3.52-3.65 (m, 26H); 3.41-3.46 (t, 2H); 3.37 (s, 3H); 2.02-2.04 (q,2H); 1.54-1.57 (m, 4H); 1.30-1.36 (m, 12H). ¹³C NMR (CDCl₃): δ=139.35,114.22, 72.06, 71.67, 70.70, 70.65, 70.18, 59.17, 33.93, 29.76, 29.67,29.60, 29.55, 29.25, 29.05, 26.21. ESI-MS: 516.5 (100%, M+1+Na⁺).

EXAMPLE 3

This example serves to illustrate photo-induced surface hydrosilylation,in accordance with embodiments of the present invention.

H-terminated silicon (100 or 111) surfaces were prepared usingprocedures similar to those described by Hines and Chidsey (Krämer etal., Chem Rev. 2003, vol. 103, p. 4367). Briefly, single-sided polishedsilicon (100) or silicon (111) wafers with a resistivity less than 5ohm·cm were cut into pieces of ca. 1×1 cm², cleaned with NH₄OH/H₂O₂/H₂O(v/v 1:1:4) at 80° C. for 20 minutes, thoroughly washed withMillipore-purified water, etched in 10% buffer-HF for 10 minutes andthen in 40% NH₄F for 10 minutes under N₂ purge, and dried with a flow ofnitrogen. The setup and procedures for photo-induced surfacehydrosilylation of H-terminated silicon substrate surfaces with alkeneswere described in detail elsewhere (Yam et al., Chem. Commun., 2004, p.2510). Briefly, a freshly prepared H—Si (100) or H—Si (111) substratewas placed inside a freshly cleaned and dried quartz cell, and tiltedwith the polished surface facing a droplet (˜1 mg) of the alkene (EG₇)that was placed on a surface in the cell. After the cell was degassed at˜0.1 mTorr for 10 minutes, the wafer was allowed to fall down onto thedroplet, sandwiching a thin and homogeneous layer of the alkene betweenthe substrate and the quartz wall. The substrate was then illuminatedwith a hand-held 254 mn UV lamp (Model UVLS-28, UVP) for 30 minutes,followed by washing sequentially with petroleum ether, ethanol, andCH₂Cl₂, and finally drying with a stream of N₂.

EXAMPLE 4

This example serves to illustrate how Si surface type can affect thestability of the resulting nanometric biomolecular array.

Resistance comparisons between the adsorption and stability ofOEG-terminated thin films on H—Si (100) and Si (111) were performedusing the method as described in the present invention. Resultsindicated that the films of α-hepta-(ethylene glycol) methyl ω-undecenylether, EG₇, on Si (111) and (100) substrates reduced adsorption ofprotein (fibrinogen) by >99%. The films were stable under a wide rangeof conditions, such as in biological buffers at pH 7.4 and 9.0 (37° C.),water (100° C.), and 2.5 M H₂SO₄ (100° C.). The films derived on Si(111) were more stable than those on Si (100). Furthermore, it wasdemonstrated that the films on Si (100) or Si (111) could be patternedby AFM anodization lithography by the method as described in the presentinvention. The resultant patterns may serve as templates for directingthe self-assembly of biomolecules such as fibrinogen, avidin, and bovineserum albumin (BSA) on the surfaces. See Yam et al., J. ColloidInterface Sci., 2005, in press.

EXAMPLE 5

This example serves to illustrate AFM anodization lithography onEG₇-coated Si (100) or Si (111) substrates, in accordance withembodiments of the present invention.

A setup for performing AFM anodization lithography on EG₇-coated Si(100) or Si (111) substrates, in accordance with embodiments of thepresent invention, is illustrated in FIG. 5. An OEG-coated silicon (100)or silicon (111) wafer was mounted on a steel sample puck using adouble-sided carbon conductive tape that was also used to attach ashort, thin Pt wire (25 μm in diameter). Another short, thin Pt wire wasconnected to the metal clip of a tip holder. The use of thin wires forelectrical connection to the wafer and tip greatly reduces the vibrationintroduced to the system. Both Pt wires were connected through a BNCcable to a digital delay/pulse generator equipped with a homemadeamplifier. The steel sample puck was mounted. onto the AFM scanner thatwas insulated with a thin parafilm. AFM anodization lithograpy wasperformed under ambient conditions with a relative humidity of 25-55%,using a silicon cantilever with a force constant of 0.3 N/m andresistivity of ˜0.08 ohm.cm. During scanning of the sample in contactmode (load: ˜1 nN; scan size: 5×5 μm; scan rate: 29.8 μm/s), bursts of10 pulses of +17 V square waves (pulse duration: 1 μs; interval betweentwo pulses: 8.33 ms; interval between two bursts: 4.29 seconds) wereapplied to the sample while the tip was grounded. The nanolithographywas completed in one scan, taking 85 seconds. For relocation of thenanopattemed areas after subsequent ex situ treatment of the sample, atiny X mark with a line width of ˜8 μm was drawn on the substrate with adiamond pen before nanolithgraphy, and the position of the cantileverrelative to the mark shown by the CCD camera of the AFM was recordedFIG. 6 illustrates the above-described AFM anodization lithography of anOEG monolayer on a Si surface, wherein the patterned (anodized) regionscomprise carboxylic acid moieties.

EXAMPLE 6

This example serves to illustrate AFM imaging, in accordance withembodiments of the present invention.

After nanolithography, the pulse generator was disconnected, andtopography and friction AFM images were simultaneously acquired incontact mode at 90° scan angle with the same tip used fornanolithography. For imaging protein-coated surfaces, a soft cantileverwith a force constant of 0.03 N/m was used at a loading force of ˜1 nN.

EXAMPLE 7

This example serves to illustrate derivatization of the nanoarrays withbiomolecules, in accordance with embodiments of the present invention.

The patterned substrates were treated sequentially with (a) a solutionof succinic anhydride (100 mg), and DMAP (12 mg) in pyridine (1 ml) for30 minutes; (b) a solution of EDAC (1 mg) and avidin (0.1 mg) in PBS (1ml) for 5 minutes; (c) BSA (1 mg) in PBS (1 ml) for 5 minutes; (d)biotin-BSA (1 mg) in PBS (1 ml) for 5 minutes; (e) avidin (0.1 mg) inPBS (1 ml) for 5 minutes; and (f) proteinase K (1 mg) in PBS (1 ml) for3 hours. All treatments were carried out under ambient conditions. Aftereach step, the substrates were thoroughly washed with Millipore water,dried with a stream of N₂, and immediately imaged by AFM.

All patents and publications referenced herein are hereby incorporatedby reference. It will be understood that certain of the above-descnrbedstructures, functions, and operations of the above-described embodimentsare not necessary to practice the present invention and are included inthe description simply for completeness of an exemplary embodiment orembodiments. In addition, it will be understood that specificstructures, functions, and operations set forth in the above-describedreferenced patents and publications can be practiced in conjunction withthe present invention, but they are not essential to its practice. It istherefore to be understood that the invention may be practiced otherwisethan as specifically described without actually departing from thespirit and scope of the present invention as defined by the appendedclaims.

1. A method comprising the steps of: a) contacting OEG-terminatedalkenes with a hydrogen-terminated Si surface to form a contactedsurface; b) photolyzing the contacted surface to effect Si—C bondingbetween the OEG-terminated alkenes and the Si surface and form aOEG-coated Si surface comprising a monolayer of OEG bound to the Sisurface through Si—C bonds; c) lithographically anodizing regions on thetop of the OEG monolayer of the OEG-coated Si surface via AFManodization lithography to yield a nanolithographically-patternedOEG-coated Si surface comprising regions with enhanced associabilitytoward biomolecules; and d) depositing at least one type of biomoleculein the regions of enhanced associability to form a nanometricbiomolecular array.
 2. The method of claim 1, wherein the OEG-terminatedalkenes comprise an EG sequence selected from the group consisting ofEG₁-EG₂₀, and combinations thereof.
 3. The method of claim 1, whereinthe OEG-terminated alkenes comprise PEG-terminated alkenes.
 4. Themethod of claim 1, wherein the Si surface is atomically flat.
 5. Themethod of claim 1, wherein the Si surface is selected from the groupconsisting of (100), (111), and combinations thereof.
 6. The method ofclaim 1, further comprising a step of washing the OEG-coated Si surfaceprior to lithographically anodizing regions on top of it.
 7. The methodof claim 1, wherein the nanolithographically-patterned regions of theOEG-coated Si surface comprise nanowells.
 8. The method of claim 1,wherein the nanolithographically-patterned regions of the OEG-coated Sisurface comprise functional moieties selected from the group consistingof carboxylic acid, aldehyde, hydroxyl, and combinations thereof.
 9. Themethod of claim 8, wherein the functional moieties provide, at least inpart, the enhanced associability toward biomolecules.
 10. The method ofclaim 1, wherein the nanolithographically-pattemed regions of theOEG-coated Si surface comprise hydroxyl moieties that can be convertedinto carboxylic acid moieties
 11. The method of claim 1, wherein the atleast one type of biomolecule is selected from the group consisting ofproteins, oligonucleotides, and combinations thereof.
 12. The method ofclaim 10, wherein the at least one type of biomolecule comprises avidin.13. The method of claim 1, wherein at least some of the at least onetype of biomolecule binds with the regions with enhanced associabilityvia amide bonds.
 14. The method of claim 1, further comprising the stepsof: a) depositing biomolecular analyte; and b) analyzing the array todetermine the regions in which the biomolecular analyte exhibits abinding affinity.
 15. The method of claim 14, further comprising a stepof regenerating the nanolithographically-patterned OEG-coated Si surfacecomprising regions with enhanced associability toward biomolecules bytreatment of said surface with proteinase K, wherein the proteinase Kserves to catalyze hydrolytic fragmentation of proteins bound to saidsurface.
 16. A nanometric biomolecular array comprising: a) a Sisubstrate; b) a monolayer of OEG bonded to the Si substrate via Si—Cbonds, wherein regions at the top of the monolayer have beenlithographically-pattemed; and c) biomolecules associated with thelithographically-patterned regions of the OEG monolayer.
 17. Thenanometric biomolecular array of claim 16, wherein the Si surface isatomically flat.
 18. The nanometric biomolecular array of claim 16,wherein the Si surface is selected from the group consisting of (100),(111), and combinations thereof.
 19. The nanometric biomolecular arrayof claim 16, wherein the OEG comprises an EG sequence selected from thegroup consisting of EG₁-EG₂₀, and combinations thereof.
 20. Thenanometric biomolecular array of claim 16, wherein the biomolecules areselected from the group consisting of proteins, oligonucleotides, andcombinations thereof.
 21. The nanometric biomolecular array of claim 16,wherein the biomolecules are associated by a bonding means selected fromthe group consisting of covalent bonding, ionic bonding, electrostaticforces, and combinations thereof.
 22. The nanometric biomolecular arrayof claim 16, wherein the biomolecules are associated with thelithographically-patterned regions of the OEG monolayer via amide bonds.23. The nanometric biomolecular array of claim 16, wherein thebiomolecules are operable for binding molecular analyte.
 24. Themanometric biomolecular array of claim 16, wherein said array isoperable for sequencing biomolecular analyte selected from the groupconsisting of oligonucleotides, proteins, and combinations thereof. 25.The nanometric biomolecular array of claim 16, wherein said array isoperable for screening drug candidates.